The aim of this proposal is to increase our fundamental knowledge of neovascularization and to determine the time course and dosage of delivery of the Vascular Endothelial Growth Factor (VEGF) required to achieve normal blood vessel development and therapeutic angiogenesis in adults. The long-term goal of this research is to develop safe and efficacious treatments for ischemic tissue in humans by the delivery of angiogenic factors. Specifically, the proposed studies will test the following hypotheses: (1) VEGF has different physiological effects on ischemic (blood-deprived) and on non-ischemic muscle. (2) VEGF has dose-dependent effects. (3) VEGF can recruit precursor cells (stem cells) to form a multi-cell type vascular tissue in adults. To address these hypotheses, regulatable VEGF gene delivery systems will be developed both to examine the physiological effects of VEGF at carefully controlled dosages and to explore their use in a clinical setting. It has been known for several years that VEGF causes new blood vessel growth in ischemic muscle, but not in non-ischemic muscle. Data from our laboratory have shown for the first time that VEGF delivered at excessively high levels via genetically engineered myoblasts can result in the formation of hemangiomas (endothelial tumors), even in non-ischemic muscle. Thus, the ability to regulate the expression of introduced angiogenic factor genes may be critical. Clearly, the type of rigorous characterization of the diverse dose-dependent physiological responses to VEGF proposed here can be extended to other angiogenic factors (e.g. the angiopoietins) in order to better understand blood vessel modeling and remodeling. Because myoblast implantation has led to the most marked physiological response to VEGF reported to date, this approach is particularly well-suited to testing the effects of regulatable promoters that allow modulation of the level and timing of expression of angiogenic factors required to ensure both safety and efficacy. Finally, since VEGF serves as an in vivo chemoattractant for circulating endothelial precursor cells, the purification and characterization of such cells is possible, which is both of fundamental interest and may lead to improved therapies for vascular healing and tissue engineering.